This proposal describes several means of investigation of the mechanisms by which changes in chromosome structure occur at the time of activation of gene expression. For this investigation we will use the giant chromosomes of Drosophila in which puffs are visible in the light microscope at sites where intense RNA synthesis occurs. We have developed an in vitro system for the activation of several puffs at the well characterized heat shock loci. This assay will be used to identify substances responsible for the puffing and to understand the mechanisms by which the local structure of the chromosome is modified leading to unwinding, production of visible puffs and transcription. In addition we propose to isolate DNA fragments representing these loci, clone them in plasmids, and elucidate their structure as a parallel biochemical approach to understanding the mechanism of gene activation. This system offers several unique advantages including the possibility of parallel cytological and biochemical investigation, in vitro activation of gene expression and coordinate control of gene expression at several unlinked loci.